MethylFlash ™ Hydroxymethylated DNA Quantification Kit
Contents
1. Note 1 For a single point control add 1 ul of HF5 at a concentration of 2 ng ul as prepared in Step 2 for the standard curve add 1 ul of Diluted HF5 at concentrations of 0 2 to 5 ng ul see the chart in Step 2 The final amounts should be 0 2 0 5 1 2 and 5 ng per well 2 For optimal binding sample DNA volume added should not exceed 8 ul 3 To ensure that the HF3 HF4 Diluted HF5 and sample DNA are completely added into the wells the pipette tip should be put into the HF2 solution in the well and aspirated in out 1 2 times Cover strip plate with plate seal or Parafilm M and incubate at 37 C for 90 min 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 10 03 P 1037 e Remove the HF2 Binding Solution from each well Wash each well with 150 ul of the Diluted HF1 1X Wash Buffer each time for three times This can be done by simply pipetting Diluted HF1 in and out of the wells 4 Hydroxy methylated DNA Capture a Dilute HF6 at 1 1000 ratio with the Diluted HF1 b Add 50 ul of the Diluted HF6 to each well then cover and incubate at room temperature for 60 min c Remove the Diluted HF6 solution from each well d Wash each well with 150 ul of the Diluted HF1 each time for three times e Dilute HF7 at 1 2000 ratio2. Incubation time is too long The incubation time at Step 3d should not exceed 2 h Over development of fluorescence Decrease the development time in Step 5b Large variation between replicate wells Fluorescent reaction is not evenly occuring due to an inconsistency in pipetting time Ensure HF9 Fluoro Developer is added at the same time between replicates or otherwise maintain a consistent timing in between each addition of solutions Fluorescent reaction is not occurring evenly due to an inconsistent order of adding solutions Ensure all solutions particularly HF9 Fluoro Developer are added in the same order each time as all other solutions The solutions are not evenly added due to an inconsistency in pipetting volume Ensure the solution in each pipette tip is equal in the multi channel pipette Equilibrate the pipette tip in any solutions before adding them Ensure the solutions especially those with small volumes e g 1 ul are completely added into the wells Solutions or antibodies were not actually added into the wells Do not allow the pipette tip to touch the outer edges or inner sides of the wells in order to prevent solutions from sticking to the surface Did not sufficiently shake the solutions in the wells after adding sample or positive control at step 3c Gently and evenly shake the plate frame across a flat surface so that the solutions in the wells a
3. MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric Base Catalog P 1037 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric is suitable for detecting global DNA hydroxymethylation status using DNA isolated from any species such as mammals plants fungi bacteria and viruses in a variety of forms including but not limited to cultured cells fresh and frozen tissues paraffin embedded tissues plasma serum samples and body fluid samples This kit is particularly suitable for samples only available in small amounts such as laser capture microdissection samples and embryos Input DNA The amount of DNA for each assay can be 20 200 ng For optimal quantification the input DNA amount should be 100 ng as hydroxymethylated DNA hmDNA is generally less than 0 6 of total DNA Starting Material Starting materials can include various tissue or cell samples such as cells from flask or microplate cultured cells fresh and frozen tissues paraffin embedded tissues plasma serum samples body fluid samples etc Internal Control Both negative and positive DNA controls are provided in this kit A standard curve can be performed range 0 2 to 5 ng or a single quantity of hydroxymethylated DNA can be used as a positive control Because global hydroxymethylation can vary from tissue to tissue and from normal and diseased states it is advised to run replicat
4. T C G T C G A C G The broader functions of 5 hmC in epigenetics are still a mystery today However a line of evidence does show that 5 hmC plays a role in DNA demethylation chromatin remodeling and gene expression regulation specifically in brain specific gene regulation 1 Conversion of 5 mC to 5 hmC greatly reduced the affinity of MBD proteins to methylated DNA 2 The observation that formation of 5 hmC by oxidative damage or by addition of aldehydes via DNMTs prevents DNMT mediated methylation of the target cytosine 3 5 hmC may recruit specific binding proteins that alter chromatin structure or DNA methylation patterns 4 5 hmC accounts for roughly 40 percent of the methylated cytosine in Purkinje cells and 10 percent in granule neurons Because of the presence of 5 hmC in DNA with unclear functions in gene regulation and the discovery of the enzymes that produce 5 hmC it is considered rather important to know the distribution of this base in different cell types and in different compartments of the genome of mammaiians It is particularly important to identify hydroxymethylation status in human cell tissues with and without diseases Several chromatography based techniques such as HPLC and TLC mass spectrometry are used for detecting 5 hmC However these methods are time consuming and have low throughput with high costs Currently used methylated DNA analysis methods including restriction enzyme digestion and bisulfite or MeDIP
5. and specificity of 5 hydroxymethylcytosine detection achieved by the MethylFlash kit Synthetic unmethylated DNA contains only cytosine methylated DNA contains only 5 methylcytosine and hydroxymethylated DNA standard contains only 5 hydroxymethylcytosine were added into the assay wells at different concentrations and then measured with the MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input DNA Amount DNA amount can range from 20 ng to 200 ng per reaction An optimal amount is 100 ng per reaction Starting DNA may be in water or in a buffer such as TE DNA Isolation You can use your method of choice for DNA isolation Epigentek offers a series of genomic DNA isolation kits for your convenience 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 5 Printed 2014 10 03 P 1037 DNA Storage Isolated genomic DNA can be stored at 4 C short term or 20 C long term until use 1 Preparation of 1X Wash Buffer HF1 48 Assay Kit Add 13 ml of HF1 10X Wash Buffer to 117 ml of distilled water pH 7 2 7 5 96 Assay Kit Add 26 ml of HF1 10X Wash Buffer to 234 ml of distilled water pH 7 2 7 5 N
6. correctly Insufficient input materials Ensure that a sufficient amount of positive control gt 1 ng and samples gt 100 ng is added into the wells 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 9 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 10 03 Epigentek Group Inc All rights reserved Products are for research use only P 1037 Incorrect fluorescence reading Check if appropriate fluorescence wavelength 530 ex 590em nm is used Kit was not stored or handled properly Ensure all components of the kit were stored at the appropriate temperature and the cap is tightly capped after each opening or use No signal or weak signal in only the positive control wells The positive control DNA is insufficiently added to the well in Step 3c Ensure a sufficient amount of positive control DNA is added The HF5 Positive Control is degraded due to improper storage conditions Follow the Shipping amp Storage guidance in this User Guide for storage of HF5 Positive Control High background present in the negative control wells Insufficient washing of wells Check if washing recommendations at each step is performed according to the protocol Contaminated by sample or positive control DNA Ensure the well is not contaminated from adding sample or positive control DNA accidentally or from using contaminated tips
7. emission 590 O 1 5 ml microcentrifuge tubes Page 2 Printed 2014 10 03 P 1037 Incubator for 37 C incubation Plate seal or Parafilm M Distilled water 1 X TE buffer pH 7 5 to 8 0 1 X PBS pH 7 2 to 7 5 Isolated DNA of interest OoOdaqacqaqgdo GENERAL PRODUCT INFORMATION Quality Control Each lot of the MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The MethylFla
8. well plate setup for standard curve preparation in a 48 assay format in a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate Well Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A HF3 HF3 Sample Sample Sample Sample B HF4 HF4 Sample Sample Sample Sample c HF5 0 2 ng ul HF5 0 2 ng ul Sample Sample Sample Sample D HF5 0 5 ng pl HF5 0 5 ng l Sample Sample Sample Sample E HF5 1 ng ul HF5 1 ng ul Sample Sample Sample Sample F HF5 5 ng ul HF5 5 ng ul Sample Sample Sample Sample G HF5 10 ng ul HF5 10 ng ul Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample TROUBLESHOOTING Problem Possible Causes Suggestions No signals for both the Reagents are added incorrectly Check if reagents are added in the proper positive control and order and if any steps in the protocol may samples have been omitted by mistake The well is incorrectly washed Ensure the well is not washed before before DNA binding adding positve control and samples The bottom of the wells are not Ensure the solution coats the bottom of compeleted covered by the HF2 the well by gently tilting from side to side Binding Solution or shaking the plate several times Incubation time and temperature Ensure the incubation time and are incorrect temperature described in the protocol are followed
9. With Frame 6 12 47 User Guide 1 1 RT Spin the solution down to the bottom prior to use Note The HF3 Negative Control is an unmethylated polynucleotide containing 20 of cytosine The HF4 Negative Control II is a methylated polynucleotide containing 20 of 5 methylcytosine The HF5 Positive Control is a hydroxymethylated polynucleotide containing 20 of hydroxymethylcytosine SHIPPING amp STORAGE The kit is shipped in three parts the first part at ambient room temperature and the second and third parts on frozen ice packs at 4 C Upon receipt 1 Store HF3 HF4 HF5 HF7 HF8 and HF9 at 20 C away from light 2 Store HF1 HF6 HF10 and 8 Well Assay Strips at 4 C away from light 3 Store HF2 and HF11 at room temperature away from light Note Check if wash buffer HF1 contains salt precipitates before using If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved All components of the kit are stable for 6 months from date of shipment when stored properly MATERIALS REQUIRED BUT NOT SUPPLIED 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only O Adjustable pipette O Aerosol resistant pipette tips O Fluorescence microplate reader capable of reading fluorescence at excitation 530 and
10. e for optimal slope calculation Now calculate the amount and percentage of 5 hmC in your total DNA using the following formulas Sample RFU HF4 RFU Slope x 5 5 hmC ng 5 hmC Amount ng 5 hmC X 100 S S is the amount of input sample DNA in ng 5 is a factor to normalize 5 hmC in the positive control to 100 as the positive control contains only 20 of 5 hmC Example calculation Average RFU of HF4 is 900 Average RFU of sample is 8900 Slope is 15000 RFU ng S a L00 ng 8900 900 S hmC ng 0 107 ng 15000 x 5 0 107 5 hmC X 100 0 107 100 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 8 Printed 2014 10 03 P 1037 SUGGESTED STRIP WELL SETUP Table 1 The suggested strip well plate setup using a single point positive control in a 48 assay format in a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate Well Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A HF3 HF3 Sample Sample Sample Sample B HF4 HF4 Sample Sample Sample Sample c HF5 HF5 Sample Sample Sample Sample D Sample Sample Sample Sample Sample Sample E Sample Sample Sample Sample Sample Sample F Sample Sample Sample Sample Sample Sample G Sample Sample Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample Table 2 The suggested strip
11. e samples to ensure that the signal generated is validated This kit will allow the user to quantify an absolute amount of hydroxymethylated DNA and determine the relative hydroxymethylation states of two different DNA samples Precautions To avoid cross contamination carefully pipette the sample or solution into the strip wells Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 10 03 Epigentek Group Inc All rights reserved Products are for research use only P 1037 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 1037 48 Cat P 1037 96 Upon Receipt HF1 10X Wash Buffer 14 ml 28 ml 47 HF2 Binding Solution 5 ml 10 ml RT HF3 Negative Control 20 ug ml 10 ul 20 ul 20 C HF4 Negative Control II 20 ug ml 10 pl 20 ul 20 C HF5 Positive Control 20 ug ml 10 ul 20 ul 20 C HF6 Capture Antibody 1000 ug ml 4ul 8 ul 4 HF7 Detection Antibody 400 ug ml 8 ul 16 ul 20 C HF8 Enhancer Solution 8 ul 16 ul 20 C HF9 Fluoro Developer 8 ul 16 ul 20 C HF10 Fluoro Enhancer 8 ul 16 ul 47 HF11 Fluoro Dilutor 4ml 8 ml RT 8 Well Assay Strips
12. formula Sample RFU HF4 RFU S 5 hmC xX 100 HFS RFU HF4 RFU x 5 P 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 10 03 Epigentek Group Inc All rights reserved Products are for research use only P 1037 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only S is the amount of input sample DNA in ng P is the amount of input positive control HF5 in ng 5 is a factor to normalize 5 hmC in the positive control to 100 as the positive control contains only 20 of 5 hmC Example calculation Average RFU of HF4 is 900 Average RFU of HF5 is 30900 Average RFU of Sample is 8900 S is 100 ng P is 2 ng 8900 900 100 S hmC x 100 0 107 30900 900 x5 2 Absolute Quantification To quantify the absolute amount of hydroxymethylated DNA using an accurate calculation first generate a standard curve and plot the RFU values versus the amount of HF5 at each concentration point Next determine the slope RFU ng of the standard curve using linear regression Microsoft Excel s linear regression functions are suitable for such calculation and the most linear part at least 4 concentration points including 0 point of the standard curv
13. ge 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 10 03 Epigentek Group Inc All rights reserved Products are for research use only P 1037 PRINCIPLE amp PROCEDURE The MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric contains all reagents necessary for the quantification of global DNA hydroxymethylation In this assay DNA is bound to strip wells that are specifically treated to have a high DNA affinity The hydroxymethylated fraction of DNA is detected using capture and detection antibody and then quantified fluorometrically by reading the RFU relative fluorescence units with a fluorescence spectrophotometer The amount of hydroxymethylated DNA is proportional to the fluorescence intensity measured Prepare genomic DNA We recommend using Epigentek s series of DNA isolation kits Bind DNA to assay well Wash wells then add capture antibody Wash wells then add detection antibody and enhancer solution Add fluoro developing solution for fluorescence then measure RFU Schematic procedure of the MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric ASSAY PROTOCOL RFU 80000 70000 60000 50000 40000 30000 20000 10000 Hydroxymethylated DNA H Methylated DNA Unmethylated DNA 0 01 05 1 2 5 20 Input DNA ng Fig 1 Demonstration of high sensitivity
14. mediated MS PCR and sequencing are also not suitable for 5 hmC detection as 5 hmC and 5 mC are virtually indistinguishable with these methods To address this problem Epigentek offers the MethylFlash Hydroxymethylated DNA Quantification Kit Fluorometric which uses a unique procedure to quantify global DNA hydroxymethylation The kit has the following advantages and features e Fluorometric assay with easy to follow steps for convenience and speed The entire procedure can be finished within 3 hours and 20 minutes e High sensitivity of which detection limit can be as low as 10 pg of hydroxymethylated DNA e High specificity with no cross reactivity to unmethylated cytosine and methylcytosine Only hydroxymethylated DNA 5 hmC is detected e Universal positive and negative controls are included which are suitable for quantifying hydroxymethylated DNA from any species e Strip well microplate format makes the assay flexible manual or high throughput analysis e Simple reliable and consistent assay conditions References Robertson KD Nat Rev Genet 6 597 610 2005 Kriaucionis S et al Science 324 929 930 2009 WYATT GR et al Biochem J 55 774 8 1953 Tahiliani M et al Science 324 930 935 2009 Valinluck V et al Nucleic Acids Res 32 4100 4108 2004 Valinluck V et al Cancer Res 67 946 50 2007 Jin SG et al Nucleic Acids Res 38 e125 2010 Ni Or gt CaN 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pa
15. orometric P 1036 MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 11 Printed 2014 10 03 P 1037
16. ote This Diluted HF1 1X Wash Buffer can now be stored at 4 C for up to six months All other diluted solutions should be kept on ice at all times and should be discarded if not used within the same day 2 Preparation of Diluted Positive Control HF5 Single Point Control Preparation Dilute HF5 Positive Control with 1X TE buffer to 2 ng ul 1 ul of HF5 9 ul of TE buffer Suggested Standard Curve Preparation First dilute HF5 to 5 ng ul 3 ul of HF5 9 ul of 1X TE buffer Then further prepare five different concentrations with the 5 ng ul diluted HF5 and 1X TE into 0 2 0 5 1 0 2 0 and 5 ng ul according to the following dilution chart Resulting HF5 Tube HF5 Sng ul 1XTE Concentration 1 0 5 ul 12 0 ul 0 2 ng ul 2 1 0 ul 9 0 ul 0 5 ng ul 3 1 0 ul 4 0 ul 1 0 ng ul 4 2 0 ul 3 0 ul 2 0 ng ul 5 5 0 ul 0 0 ul 5 0 ng ul 3 DNA Binding a Predetermine the number of strip wells required for your experiment Carefully remove un needed strip wells from the plate frame and place them back in the bag seal the bag tightly and store at 4 C b Add 80 ul of HF2 Binding Solution to each well Cc d Add 1 ul of HF3 1 ul of HF4 1 ul of Diluted HF5 see note below and 100 ng of your Sample DNA 1 8 ul into the designated wells depicted in Table 1 or Table 2 Mix solution by gently tilting from side to side or shaking the plate several times Ensure the solution coats the bottom of the well evenly
17. re better distributed Do not stir 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 10 Printed 2014 10 03 P 1037 Did not use the same pipette device throughout the experiment Use the same multi channel pipette device throughout the entire experiment as different pipette devices may have slight variations in performance Capture Antibody vial appears to be empty or insufficient in volume Buffer evaporated due to the very small volumes resulting in a higher concentrated antibody Add 1X PBS buffer into the Capture Antibody vial until you restore the correct intended volume according to the Kit Contents described in this User Guide Mix and centrifuge prior to use RELATED PRODUCTS DNA Sample Preparation P 1003 FitAmp General Tissue Section DNA Isolation Kit P 1004 FitAmp Plasma Serum DNA Isolation Kit P 1006 DNA Concentrator Kit P 1007 FitAmp Gel DNA Isolation Kit P 1009 FitAmp Paraffin Tissue Section DNA Isolation Kit P 1017 FitAmp Urine DNA Isolation Kit P 1018 FitAmp Blood and Cultured Cell DNA Extraction Kit Global DNA Methylation Quantification P 1034 MethylFlash Methylated DNA Quantification Kit Colorimetric P 1035 MethylFlash Methylated DNA Quantification Kit Flu
18. sh Hydroxymethylated DNA Quantification Kit Fluorometric and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW DNA methylation occurs by the covalent addition of a methyl group at the 5 carbon of the cytosine ring resulting in 5 methylcytosine 5 mC In somatic cells 5 mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG whereas in embryonic stem ES cells a substantial amount of 5 mC is also observed in non CpG contexts The biological importance of 5 mC as amajor epigenetic modification in phenotype and gene expression has been widely recognized Quite recently a novel modified nucleotide called 5 hydroxymethyl cytosine 5 hmC has been detected to be abundant in mouse brains and embryonic stem cells 5 hydroxymethylcytosine was first seen in bacteriophages in 1952 In mammals it can be generated by the oxidation of 5 methylcytosine a reaction mediated by the Tet family of enzymes and Dnmt proteins It is a hydroxylated and methylated form of cytosine 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 10 03 Epigentek Group Inc All rights reserved Products are for research use only P 1037 yt Ht HH of N c smo hme Unmethylated DNA Methylated DNA Hydroxymethylated DNA T C G T C G A C G T C G T C G A C G
19. with the Diluted HF1 f Add 50 ul of the Diluted HF7 to each well then cover and incubate at room temperature for 30 min g Remove the Diluted HF7 solution from each well h Wash each well with 150 ul of the Diluted HF1 each time for four times i Dilute HF8 at 1 5000 ratio with the Diluted HF1 j Add 50 ul of the Diluted HF8 to each well then cover and incubate at room temperature for 30 min k Remove the Diluted HF8 solution from each well Wash each well with 150 ul of the Diluted HF1 each time for five times m Wash each well with 150 ul of 1 X PBS one time 5 Signal Detection a Prepare Fluoro Development Solution by adding 1 ul of HF9 and 1 ul of HF10 into each 500 ul of HF 11 b Add 50 ul of Fluoro Development Solution into the wells and incubate at room temperature for 1 to 4 minutes away from light The color in the standard wells containing the higher concentrations may turn pink during this period Measure and read RFU relative fluorescence units on a fluorescence microplate reader at 530 x 590ey nm If the strip well frame does not fit the microplate reader transfer the solution to a standard 96 well microplate and read the RFU on a fluorescence microplate reader at 530 590 y nm 6 5 hmC Calculation Relative Quantification To determine the relative hydroxymethylation status of two different DNA samples simple calculation for the precentage of 5 hmC in your total DNA can be carried out using the following
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